Metal–Phenolic Network-Loaded Sodium Alginate-Based Antibacterial and Antioxidant Films Incorporated with Geranium Essential Oil

Abstract

Owing to its natural degradability and excellent film-forming characteristics, sodium alginate (SA) is gaining growing popularity in the field of food packaging. However, the insufficient antioxidant and antibacterial properties hinder its application. In the current research, protocatechuic acid (PCA) and Fe³⁺ were utilized to fabricate a metal polyphenol network structure. Subsequently, geranium essential oil emulsion (GEOE) was incorporated into the SA matrix, and SA-based films were prepared through the flat-sheet casting method. The impacts of PCA/Fe and various concentrations of GEOE on the physical, structural, as well as functional characteristics of SA-based films were comprehensively examined. The thickness of the prepared SA-based films was between 30 and 50 μm. The results showed that PCA/Fe, GEOE, and SA exhibited good biocompatibility, and the formed films were uniform. The incorporation of PCA/Fe and GEOE significantly improved the UV blocking ability, thermal stability, and antibacterial activity of SA-based films. In addition, PCA/Fe and GEOE enhanced the total antioxidant capacity of SA-based films from 3.5% to 88%. This research could provide some theoretical basis for the utilization of metal polyphenol networks and natural essential oils within the realm of food active packaging films.

1. Introduction

With the increasing prominence of environmental issues and the call for sustainable development, academia and industry are paying more attention to environmentally friendly biodegradable plastics [1,2]. Traditional plastics synthesized from petroleum resources have a serious impact on the environment, and their difficulty in degrading leads to continued pollution of the ecosystem [3]. Therefore, exploring biopolymer materials extracted from natural resources that are environmentally friendly and biodegradable has become an urgent need.

Food polymers, like natural proteins, polysaccharides, and polyphenols, have been extensively applied in the food industry, medicine, and packaging [4]. These natural polymers have abundant sources, biodegradability, and good film-forming properties and could be used to prepare a variety of edible films [4]. Natural polymers like chitosan, carrageenan, SA, and soybean protein isolate show the advantages of being biodegradable, safe, and non-toxic and having good film-forming properties, making them viable alternatives to packaging films [5]. Sodium alginate (SA), a polyanionic polysaccharide carbohydrate, is derived from brown algae and consists of a combination of β-D-mannuronic acid (M) and α-L-guluronic acid (G) [6]. The advantages of SA in active food packaging films mainly lie in its good film-forming properties, safety, biodegradability, and controllable air permeability. It is especially suitable for food packaging applications with high requirements for freshness and safety. Although SA shows biodegradability, biocompatibility, and good film-forming properties, it still has some limitations like strong hydrophilicity, thermal instability, poor mechanical properties, and antioxidant activity [7]. Therefore, the addition of other active substances to ameliorate these defects might help to extend its application.

In recent years, emerging metal–phenolic networks (MPNs) that are mainly composed of metal ions and phenolic ligands chelated through coordination bonds have attracted much attention due to their fast assembly speed and high mechanical and thermal stability [8,9]. Furthermore, this method is highly customizable on account of the diversity and functional variety of metal elements and polyphenol molecules. Usually, the catechol or galloyl group of phenolic compounds serves as an important chelating site for metal ions, and phenolic compounds have good antioxidant activity, which could well make up for the low antioxidant activity of SA [10]. Meanwhile, MPN shows good adhesion properties and could enhance the adhesion ability of materials [11]. Moreover, the inorganic–organic hybrid characteristic of MPN enhances its compatibility with the polymer matrix, rendering it appropriate for being employed as a filler in the preparation of high-performance functional films [12]. Yun et al. [13] reported that incorporating Fe³⁺-tannic acid composite into films could greatly improve their mechanical properties and stability. Mao et al. [10] added EGCG-Fe to SA-based films, introduced thymol essential oil, reported a significant improvement in the mechanical strength, thermal stability, and UV blocking capabilities of SA-based films, and enhanced the antibacterial and antioxidant properties. Protocatechuic acid (PCA) is a water-soluble phenolic acid substance widely existing in various edible plants, including white grapes, green tea, olives, etc., and it is also the main metabolite of dietary anthocyanins [14]. PCA has a variety of biological activities, such as antibacterial, anti-hyperglycemia, anti-inflammatory, and antioxidant activities [15]. However, to the best of our knowledge, few applications of PCA-Fe in food packaging films have been reported up to now. Studying the metal polyphenol networks of novel polyphenol compounds reacting with metal ions will contribute to the wide application of natural polyphenol compounds in food, materials, and other fields.

In addition, the antibacterial activity of pure SA film is relatively weak, which will hinder its multiple applications in food packaging. Many natural plant essential oils are recognized as “Generally Recognized as Safe” (GRAS) natural antibacterial agents, possess good antioxidant and antibacterial activities, and have broad potential applications in food antibacterial packaging [16]. Once essential oils were incorporated into the SA film, the film was able to effectively impede food oxidation and suppress microbial growth. This demonstrated that the newly developed film exhibited a favorable preservation effect. At present, there are extensive studies on the antibacterial films of natural plant essential oils, including thyme essential oil [10], oregano essential oil [17,18], cinnamon essential oil [19], etc. These essential oils exhibit good antibacterial and antioxidant properties and play an important role in films in food packaging [20,21]. However, there is still a lack of application of geranium essential oil (GEO) in food packaging films. Furthermore, there are few studies on using metal polyphenol networks to enhance the antioxidant activity of food packaging films. We hypothesized that combining metal polyphenol networks with GEO and incorporating them into SA film might result in better antioxidant and antibacterial effects.

Consequently, the primary objective of the current research is to fabricate a novel antioxidant packaging material by incorporating the metal polyphenol network into the SA-based film. Meanwhile, GEO with antibacterial effects was added to enhance the antibacterial properties of the film. The effects of MPN and GEO concentrations on the physical and chemical properties of the SA-based film such as the apparent structure, thermal stability, barrier capacity, and UV shielding ability were systematically explored. Additionally, the antioxidant and antibacterial activities of the SA-based films were explored. The present study might provide a certain theoretical reference for the application of metal polyphenol networks and natural plant essential oils in polysaccharide-based food active packaging films.

2. Materials and Methods

2.1. Materials

Sodium alginate (SA, with a viscosity of 200 ± 20 mPa.s) was purchased from Yuanye Biochemical Technology Co., Ltd. (Shanghai, China). Iron (III) chloride hexahydrate (FeCl₃·6H₂O), protocatechuic acid (PCA) was bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). FITC and Nile red were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). PBS buffer solutions were purchased from Macklin (Shanghai, China). Geranium essential oil was bought from Yuanye Biochemical Technology Co., Ltd. (Shanghai, China) and it mainly contains ingredients such as citronellol, geraniol, linalool, menthone, limonene, isomenthone, etc. 2,2′-azinobis-(3-ethylbenzothiazoline)-6 sulphonic acid (ABTS), Luria-Bertani (LB) medium, and agar were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other reagents utilized in this study were of analytical grade and were purchased from Guoyao Chemical Co., Ltd. (Shanghai, China).

2.2. Fabrication of Geranium Essential Oil Emulsion

The different amounts of geranium essential oil (GEO) and 0.8 mL Tween 80 were added into 20 mL ultrapure water to prepare different solutions of GEO in water (2%, 4%, and 6%, v/v respectively) and were fully dispersed with constant stirring at a speed of 1000 rpm for 30 min. Then, the mixed solution was processed using a high-speed homogenizer (T25 Digital Ultra Turrax, IKA, Staufen im Breisga, Germany) at 10,000 rpm for 2 min to obtain the emulsion of geranium essential oil (GEOE) [10].

2.3. Film Preparation

The preparation process of the SA-based films was slightly modified compared to that in previous studies [10]. In brief, 1.5 mL PCA (10 mg/mL) was first added to the 3 mL PBS (pH 7.2) buffer solution, and then 1.5 mL ferric chloride solution (10 mg/mL) was added to it and vortexed for 30 s. The obtained mixed solution was added into 56 mL SA solution (2%, w/v) and stirred for 30 min. Then, 14 mL of different concentrations of GEOE (2%, 4%, and 6%, v/v) and 30% glycerol (based on the weight of SA) were subsequently added to the above solution and stirred continuously for 30 min. Then, the film-forming solution was sonicated for 3 min to remove the air bubbles. Finally, the film-forming solutions were poured into the Petri dish, dried in a 40 °C oven, stored in an environment with a relative humidity of 53% to balance moisture, and kept for further analysis.

2.4. Characterization of the Film

2.4.1. Thickness and Water Content

The thickness of the SA-based film was measured by a digital micrometer, and five points on the film were randomly taken and averaged after the measurement [3]. The water content determination was slightly modified from the previous study [22]. The SA-based film was cut into strips and accurately weighed. The primary weight of the SA-based film was labeled as W1. This procedure repeatedly dried the films to a constant weight in a 105 °C oven. After fully cooling for 30 min, the final weight of the film was labeled as W2. The water content was calculated using the formula below:

$$Water content\left(\%\right)={\displaystyle \frac{(W1-W2)}{\mathrm{W}1}}\times 100\%$$

2.4.2. The Distribution of Geranium Essential Oil

The prepared geranium essential oil emulsion was stained with Nile red [23]. In total, 20 μL of Nile red (1 mg/mL) was first added to 1 mL of different concentrations of GEOE, mixed, and allowed to stain for 2 h, and then the solution was observed by an inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan).

2.4.3. Scanning Electron Microscopy

The surface and cross-sectional microstructures of the SA-based film were examined via a scanning electron microscope (SEM, SU-8010, Hitachi Ltd., Tokyo, Japan) under an accelerating voltage of 5.0 kV [24]. Small pieces were cut from the SA-based film, which were then fastened onto the sample stage using double-sided conductive adhesive. Subsequently, the sample was placed inside the electron microscope for imaging to observe the microstructure of the SA-based film. All the images were captured using the XT microscope control software. In addition, the surface of the film was scanned by energy disperse spectroscopy (EDS) to determine the presence of iron ions.

2.4.4. Fourier Transform Infrared

The attenuated total reflectance FT-IR spectra of the SA-based films was determined by FT-IR (iS50, Nicolet, Thermo Scientific, Waltham, MA, USA) in the range of 4000–500 cm⁻¹ with 32 scans, according to our previous method [25].

2.4.5. X-Ray Diffraction

The X-ray diffraction (XRD) patterns of various SA-based films were determined using an X-ray diffractometer (D8 Advance, Bruker AXS Model, Karlsruhe, Germany) with Cu Kα radiation operating at a voltage of 40.0 kV. The diffraction angle was scanned within the range from 4° to 50° at a scanning speed of 1° per minute [26].

2.4.6. Color

The color change of the samples was measured by a color differential meter [27]. The instrument was used to record the color difference (represented by L*, a*, and b* values) between the film and the standard board.

2.4.7. Ultraviolet–Visible Analysis

The transmittance of the SA-based films in the wavelength range of 200 nm to 600 nm was measured using a UV–vis spectrophotometer (TU-1900, Beijing, China) [3]. The film was cut into strips sized 1 cm × 4 cm and placed into a colorimetric dish for the test.

2.4.8. Thermal Gravimetric Analysis

The thermal stability of the SA-based film samples was determined using a thermogravimetric analyzer (TA-Q500, TA Instruments, New Castle, DE, USA) [28]. Around 5 mg of the sample was placed in an aluminum crucible and scanned under a nitrogen atmosphere. The temperature was raised from 50 °C to 600 °C at a heating rate of 10 °C per minute.

2.4.9. Mechanical Properties

The mechanical properties of the SA-based films, sized 1 cm × 4 cm, were measured using a texture analyzer (Universal TA, Shanghai Tengba Instrument Technology Co., Ltd., Shanghai, China). The testing followed the ASTM D882-02 method with certain adjustments. The initial experimental distance was set at 20 mm, and the pulling speed was 10 mm/s. The tensile strength (TS) and elongation at break (EB) of the film samples were noted down.

2.5. Functional Properties of the Films

2.5.1. Antioxidant Activity

The antioxidant activity of the SA-based film specimens was measured by an ABTS free radical scavenging assay [10]. The different groups of film specimens were mixed with ABTS working solution at 37 °C in darkness for 30 min, and the absorbance at 734 nm was measured under the microplate reader (Synergy H1, BioTek, Swindon, UK). Free radical scavenging capacity was calculated using the following formula:

$$Radical scavenging ability\left(\%\right)=(1-{\displaystyle \frac{A}{A_0}})\times 100\%$$

where A and A₀ are the absorbency of the solutions with and without films, respectively.

2.5.2. Antibacterial Activity

The antibacterial activity of the SA-based film specimens was assessed by the filter sheet method [29]. In brief, the filter paper (7 mm) was soaked in different film-forming solutions. The Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacterial suspension were diluted with sterile saline to a concentration of 10⁶ CFU/mL and coated to Luria-Bertani (LB) medium, and different filter sheets were placed into petri dishes for quantitative antimicrobial coil size after 24 h in a 37 °C incubator.

2.5.3. Hemolysis Properties

The hemocompatibility of the SA-based film was evaluated following a previous report with slight modifications [29]. Briefly, 1 mL of fresh mouse whole blood was thoroughly mixed with 9 mL of PBS buffer solution. Subsequently, 0.1 mL of erythrocytes was added separately to 0.5 mL of the extracts from SA and the SA-based films of each group. Meanwhile, 0.5 mL of PBS buffer solution (pH 7.2) was added as the positive control, and 0.5 mL of 0.1% Triton X-100 solution served as the negative control. The samples of SA and the SA-based films were incubated at 37 °C for 1 h and then centrifuged at 2000 revolutions per minute (rpm) for 10 min. A microplate reader (Synergy H1, BioTek, UK) was employed to measure the absorbance of the supernatant of each sample group at a detection wavelength of 540 nm. The hemolysis rate was calculated using the following formula:

$$Hemolysis\left(\%\right)={\displaystyle \frac{(A_i-A_j)}{(A_0-A_j)}}\times 100\%$$

where A_i, A_j, and A₀ are the absorbance value of the sample, the positive control PBS, and the negative control Triton X-100, respectively.

2.6. Statistical Analysis

All the experiments were conducted in triplicate, and the data were presented as the means ± standard deviation (n = 3). Analysis of variance (ANOVA) was performed using Duncan’s test via SPSS software, version 21.0 (IBM software, Chicago, IL, USA). The significance level was set at p < 0.05.

3. Results and Discussion

3.1. Thickness and Water Content

SA-PCA/Fe-GEOE films with different GEOE addition amounts were prepared by flat-plate casting method (Figure 1).

Table 1. The thickness, water content, and antibacterial properties of different SA-based films.

Samples	Thickness (μm)	Water Content (%)	S. aureus Inhibition Zone (mm)	E. coli Inhibition Zone (mm)
SA	31.60 ± 3.51 d	14.58 ± 0.38 a	-	-
SA-PCA/Fe	38.20 ± 0.84 c	11.99 ± 1.39 a	8.33 ± 0.15 b	6.93 ± 0.15 b
SA-PCA/Fe-GEOE2	40.20 ± 0.84 c	7.28 ± 2.17 b	9.10 ± 0.20 b	7.67 ± 0.15 b
SA-PCA/Fe-GEOE4	44.60 ± 2.30 b	8.94 ± 0.81 b	9.53 ± 0.15 a	8.83 ± 0.35 a
SA-PCA/Fe-GEOE6	49.60 ± 1.52 a	7.75 ± 1.52 b	10.13 ± 0.31 a	9.50 ± 0.26 a

The results are presented as the mean ± SD in triplicate. Values in the same column with different letters are significantly different (p < 0.05). - means no inhibiting effect.

lists the effects of PCA/Fe and GEO on the thickness and water content of the SA-based film. The thickness of each group of films was between 31 and 50 μm. The thickness of the SA film in the control group was about 31.60 μm. After adding PCA/Fe and GEOE, the thickness of the film increased significantly with the concentration of GEOE. This was mainly due to the change in film thickness caused by the increase in solid dry matter [3]. Food packaging materials need to effectively block moisture to keep food quality from being affected. Relative humidity is a critical parameter in food packaging, affecting the shelf life of the product. Therefore, it is crucial to detect the moisture content of the films to understand the material interactions and the physicochemical properties of films. In this study, it was observed that PCA/Fe and GEOE showed a significant impact on the SA-based films. Compared with the control group, PCA/Fe could reduce the water content of the SA film, and with the addition of GEOE, the water content of the SA film further decreased but showed no concentration dependence. The decrease in moisture content might be attributed to the high hydrophobicity of essential oils, which further hindered contact between water and the film substrate [10]. In addition, PCA/Fe might interact with SA molecules, preventing the hydroxyl groups in SA from combining with water molecules, resulting in a reduction in the water absorption rate of the films.

3.2. The Distribution of GEO

An inverted fluorescence microscope was used to observe the distribution of GEO in the emulsions, and Nile red was used to stain GEO. Under the microscope, the tiny droplets or clumps formed by essential oils could be clearly observed (Figure S1). These droplets or clumps appeared in a relatively uniform spherical shape and were evenly distributed in different positions in the emulsions. GEO could be dyed red by Nile red. 2% GEO showed less red distribution. As the GEO concentration increased, the proportion of red gradually increased.

3.3. SEM

The surface and cross-section of the SA-based film are shown in Figure 2. At lower magnifications, the surface of each film was relatively smooth. At higher magnifications, small particles appeared on the surface of the SA film. After adding PCA/Fe and GEOE, the surface of the film became smoother, indicating that SA, PCA/Fe, and GEOE exhibited good biocompatibility [10]. Meanwhile, with the addition of GEOE, the distribution traces of essential oils on the film surface could be observed in the picture. Further observation of the microstructure of the SA-based film cross-sections also revealed a similar situation. A dense essential oil distribution could be observed on the SA film added with GEOE. In addition, we conducted EDS on the SA-PCA/Fe film surface to determine its element distribution (Figure 2), and the results showed the presence of Fe, Na, and Cl. These results suggested that PCA/Fe was successfully incorporated into the SA film.

3.4. FTIR

FTIR could be used to analyze the chemical composition of the film, including the polymer type, additives, fillers, etc. [3]. The FTIR spectra of different SA-based films are shown in Figure 3A. In the SA spectrum, the characteristic peaks of the carboxyl group usually appear around 1600 cm⁻¹ (symmetric stretching vibration) and around 1400 cm⁻¹ (asymmetric stretching vibration) [10]. In the SA-PCA/Fe spectrum, these peaks have shifted and weakened to a certain extent. This change usually indicates that the iron ions have coordinated with the carboxyl groups in sodium alginate to form a metal complex. The combination of iron ions and carboxyl groups affects the vibration frequency of the carboxyl group, causing the position of its characteristic peak to change. Compared with the SA film, the O-H stretching vibration peak (3200–3700 cm⁻¹) of the film after adding PCA/Fe is broader and stronger, indicating that there are more hydrogen bonds in the composite material. This may be because the hydrogen bonding between PCA and SA enhances the stretching vibration of O-H. Any changes in this peak indicated changes in the number of free amino groups or hydroxyl groups [22]. Compared with the pure SA film, this peak was enhanced after adding GEOE, which may be due to the increase in the number of free hydroxyl groups caused by the addition of GEOE. The peak enhancements at 2850 and 2900 cm⁻¹ appeared in the SA-PCA/Fe-GEOE film, which may be due to the stretching vibration of the C-H bonds in the methyl group [10]. These groups existed in plant essential oils, which confirmed the presence of essential oils in the sample.

3.5. XRD

XRD can be employed to uncover the crystal structures of materials and holds significant importance in the research of polysaccharide-based films [30]. Figure 3B presents the XRD patterns of various SA-based films. The pure SA film displayed an amorphous semi-crystalline state, which was in line with previous studies on polysaccharide-based films [7]. After the incorporation of PCA/Fe and different concentrations of GEOE, no new peaks emerged in the spectra of each sample group. This suggested that PCA/Fe and GEOE were uniformly dispersed in the SA-based film and had excellent biocompatibility [10]. Meanwhile, as GEOE was added, the peak intensity in the sample increased. This might be attributed to the hydrogen-bonding interaction between GEOE and SA.

3.6. Color

The effects of PCA/Fe and GEO on the color of SA film are shown in

Table 2. The color parameters of different SA-based films.

Samples	L*	a*	b*
SA	36.34 ± 5.08 a	0.33 ± 0.12 a	−0.28 ± 0.06 a
SA-PCA/Fe	15.42 ± 2.81 b	0.27 ± 0.05 a	−1.64 ± 0.19 b
SA-PCA/Fe-GEOE2	15.44 ± 1.44 b	0.18 ± 0.09 b	−1.67 ± 0.10 b
SA-PCA/Fe-GEOE4	15.53 ± 0.51 b	0.31 ± 0.03 a	−1.68 ± 0.07 b
SA-PCA/Fe-GEOE6	16.51 ± 1.19 b	0.29 ± 0.01 a	−1.46 ± 0.04 b

Results are presented as the mean ± SD in triplicate. Values in the same column with different letters are significantly different (p < 0.05).

. L* mainly reflects the brightness of the sample color, the a* value reflects the red–green degree of the sample color, and the b* value represents the yellow–blue degree of the sample color. The color of pure SA film was colorless and transparent, so its L* value was the highest, and the a* and b* values were low. When PCA/Fe was added, the L* value of the SA-PCA/Fe film decreased, the b* value increased, and the a* value did not change significantly, which indicated that PCA/Fe was evenly dispersed in the SA film, and its color was also integrated into the SA film, which increased its b* value. When GEOE was added, its L*, a*, and b* values did not change significantly, indicating that GEO essential oil had no effect on the color of the film. This was due to the essential oil itself being colorless and transparent, so it would not affect the color change.

3.7. UV

The anti-UV barrier properties in food packaging films are crucial, as they could effectively protect food from UV irradiation, slow down lipid oxidation, and extend its shelf life [21]. To further study the effects of PCA/Fe and GEOE on the optical properties of SA-based films, the UV spectra of SA-PCA/Fe and SA-PCA/Fe-GEOE films were measured, and the results are shown in Figure 3C. Compared with the pure SA film, the addition of PCA/Fe reduced the UV transmittance of the SA film, indicating that the anti-UV ability of the film was enhanced, which may be attributed to the good UV absorption ability of PCA [31]. Meanwhile, with the addition of GEOE, the UV blocking capabilities of each group of samples were significantly improved. This phenomenon might be attributed to the dense distribution of emulsion droplets in the film, which blocked the light path or scatters light [32]. When a high concentration of GEOE (6%, v/v) was added, the UV blocking ability of the film decreased. This suggested that the addition of an appropriate amount of GEOE could enhance the UV blocking ability of SA film. Mao et al. [10] also reported that thyme essential oil could improve the UV blocking ability of SA-based composite films.

3.8. TGA

TGA can be used to determine the thermal stability and thermal decomposition characteristics of the film materials [33]. The TGA and DTG curves of different SA-based films are shown in Figure 4. There were two main stages of mass loss of pure SA films during the heating process. The first stage was at 60–100 °C, which was caused by the evaporation of water in the film [8]. The second stage occurred at 175–300 °C, which was mainly caused by the thermal depolymerization of glycerol and SA [3]. It can be seen in Figure 4b that the addition of GEOE slightly reduced the initial decomposition temperature of the SA membrane, but the difference was not significant. In the SA-PCA/Fe-GEOE film, the third stage of weight loss occurred, mainly at 330–380 °C, which might be related to the addition of GEOE. This indicated that the addition of GEOE significantly enhanced the thermal decomposition temperature of SA-based films and might be explained by the interaction between PCA/Fe and SA and the high thermal stability of GEOE. In the DTG graph, the decomposition peaks of the composite film were significantly separated compared to those of the SA film, showing multiple decomposition peaks. This was mainly due to the different thermal decomposition behaviors of different components in the film and also reflected the influence of multiple cross-linked structures (such as hydrogen bonds and coordination bonds) in the composite material on the thermal decomposition process, making the decomposition process no longer single. In addition, GEOE could reduce the water content in the SA-based film and interact with the film substrate, thereby enhancing the thermal stability of the SA film. Therefore, these results suggested that PCA/Fe and GEOE could improve the thermal stability of SA-based films.

3.9. Mechanical Properties

The mechanical characteristics of a film influence its capacity to safeguard food during transportation, storage, and processing. The most frequently used indicators for assessing its mechanical performance are tensile strength (TS) and elongation at break (EB). These indicators rely on the structure and interactions of different components within the film and can be determined through tensile testing [10]. As depicted in Figure 5a,b, the TS and EB of the pure SA film were 34.48 MPa and 8.38%, respectively. Incorporating PCA/Fe notably enhanced the mechanical strength and flexibility of the SA-based films. This improvement might be attributed to the robust mechanical properties of PCA/Fe [13]. When compared with the SA-PCA/Fe film, adding GEOE led to a slight reduction in the film’s TS. This could be because GEOE weakened the molecular interactions among the polymer chains in the film, causing a loss of cohesion and mechanical resistance in the film [34]. The decline in the TS of the films after adding GEOE in this research was comparable to the findings in a study on chitosan films containing essential oils [23]. Conversely, as the GEOE concentration increased, the EB of the film rose sharply from 12.24% to 15.64%. This may result from the plasticizing effect of GEOE. The plasticizing effect can break secondary bonds and enhance the flexibility of the polymer molecular chains in the films, thus improving the film’s elongation [35].

3.10. Antioxidant Activity

In food packaging films, antioxidant activity is crucial for food preservation. The antioxidant capacity of SA-based films was measured by the ABTS radical scavenging method. It was found that the antioxidant capacity of the pure SA film was quite weak (Figure 6a). When PCA/Fe was added, the antioxidant capacity of the film was significantly increased. This phenomenon might be explained by the fact that PCA could reduce free radicals through an aromatic structure and a single electron donor. In addition, Mao et al. [8] showed that adding PCA/Fe to SA hydrogel could also improve its antioxidant activity. Meanwhile, the antioxidant activity of SA-PCA/Fe was further enhanced with the addition of GEOE, which may be due to GEOE possessing a strong hydrogen atom transfer effect and a single electron transfer effect to scavenge free radicals [36]. The above results indicated that the addition of PCA/Fe and GEOE could significantly enhance the antioxidant activity of SA-based films.

3.11. Antibacterial Activity

The antibacterial ability of food packaging films is crucial because it could effectively inhibit the growth of pathogenic microorganisms and prevent food spoilage and deterioration [32]. The inhibition zone method was used to evaluate the inhibitory effect of SA-based films on E. coli and S. aureus. The size of the inhibition zone could reflect the ability of the substance to inhibit bacteria. The pure SA film showed no inhibitory effect on E. coli and S. aureus; when PCA/Fe was added, the film exhibited a certain inhibitory effect on E. coli and S. aureus (Table 1). Song et al. [14] have reported that PCA exhibited a certain antibacterial ability, which was similar to our results. Meanwhile, with the addition of GEOE, the antibacterial ability of the film was further improved. This may be due to the effective antibacterial activity of GEO and the synergistic effect with PCA that enhanced the antibacterial activity of the film. Mao et al. [10] prepared SA films using thyme essential oil and EGCG/Fe and also discovered the synergistic antibacterial effect of the thyme essential oil and EGCG/Fe. These results indicated that the introduction of PCA/Fe and GEOE enhanced the antibacterial ability of SA-based films.

3.12. Hemolysis

Hemolysis refers to the ability of the material to dissolve red blood cells when in contact with blood [37]. The release of hemoglobin might affect food safety and quality. Therefore, during the design and evaluation process of food packaging films, its hemolytic properties need to be considered to ensure that the film materials are harmless to the human body. Hemolytic tests on SA-based films were conducted using mouse red blood cells (Figure 6b,c). The results showed that pure SA films were not hemolytic, and the introduction of PCA/Fe exerted no significant effect on the hemolysis of SA-based films. When GEOE was added, hemolysis gradually occurred and became stronger as the GEOE concentration increased. The hemolysis rate was lower when 2% GEOE was added. This might be attributed to the GEO’s dissolution of red blood cells, leading to hemolysis. These results indicated that a moderate amount of essential oil addition could prepare the films with low hemolysis rate.

4. Conclusions

Food packaging film with antioxidant, antibacterial, and hemolysis capabilities were successfully prepared through PCA/Fe and GEOE to SA film. The effects of PCA/Fe and different concentrations of GEOE on the physical, structural, and functional properties of SA-based films were comprehensively analyzed. SEM and XRD results showed that the film matrix exhibited good compatibility with the added PCA/Fe and GEOE. The addition of PCA/Fe and GEOE could enhance the UV blocking ability and thermal stability of SA-based films. Antioxidant and antibacterial analysis suggested that PCA/Fe and GEOE significantly enhanced the antioxidant and antibacterial activities of SA-based films. This study showed that adding PCA/Fe and GEOE to SA-based films possessed certain application prospects, and it will provide a certain theoretical reference for future active food packaging films.